Literature context: Fisher Scientific CAT# A-22287; RRID:AB_2620155 Protease Inhibitor Cocktail (ED
Dendritic filopodia of developing neurons function as environmental sensors, regulating the spatial organization of dendrites and proper targeting to presynaptic partners. Dendritic filopodia morphology is determined by the balance of F-actin assembled via two major nucleating pathways, the ARP2/3 complex and formins. The inverse-BAR protein MTSS1 is highly expressed in Purkinje cells (PCs) and has been shown to upregulate ARP2/3 activity. PCs in MTSS1 conditional knockout mice showed dendrite hypoplasia due to excessive contact-induced retraction during development. This phenotype was concomitant with elongated dendritic filopodia and was phenocopied by overactivation of the actin nucleator formin DAAM1 localized in the tips of PC dendritic protrusions. Cell biology assays including single-molecule speckle microscopy demonstrated that MTSS1's C terminus binds to DAAM1 and paused DAAM1-mediated F-actin polymerization. Thus, MTSS1 plays a dual role as a formin inhibitor and ARP2/3 activator in dendritic filopodia, determining final neuronal morphology.
Literature context: RRID:AB_2620155 Rat monoclonal E-cadherin Clone
The regulation of metastatic organotropism in pancreatic ductal a denocarcinoma (PDAC) remains poorly understood. We demonstrate, using multiple mouse models, that liver and lung metastatic organotropism is dependent upon p120catenin (p120ctn)-mediated epithelial identity. Mono-allelic p120ctn loss accelerates KrasG12D-driven pancreatic cancer formation and liver metastasis. Importantly, one p120ctn allele is sufficient for E-CADHERIN-mediated cell adhesion. By contrast, cells with bi-allelic p120ctn loss demonstrate marked lung organotropism; however, rescue with p120ctn isoform 1A restores liver metastasis. In a p120ctn-independent PDAC model, mosaic loss of E-CADHERIN expression reveals selective pressure for E-CADHERIN-positive liver metastasis and E-CADHERIN-negative lung metastasis. Furthermore, human PDAC and liver metastases support the premise that liver metastases exhibit predominantly epithelial characteristics. RNA-seq demonstrates differential induction of pathways associated with metastasis and epithelial-to-mesenchymal transition in p120ctn-deficient versus p120ctn-wild-type cells. Taken together, P120CTN and E-CADHERIN mediated epithelial plasticity is an addition to the conceptual framework underlying metastatic organotropism in pancreatic cancer.
Literature context: isher Scientific, Carlsbad, CA, RRID:AB_2620155).
The orientation of hair bundles on top of sensory hair cells (HCs) in neuromasts of the lateral line system allows fish to detect direction of water flow. Each neuromast shows hair bundles arranged in two opposing directions and each afferent neuron innervates only HCs of the same orientation. Previously, we showed that this opposition is established by expression of Emx2 in half of the HCs, where it mediates hair bundle reversal (
Literature context: hermo Fisher Scientific A22287, RRID:AB_2620155 Goat polyclonal anti-rat IgG (H
Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.
Literature context: Fisher Scientific Cat# A22287; RRID:AB_2620155 Chemicals, Peptides, and Recomb
The differentiation of alveolar epithelial type I (AT1) and type II (AT2) cells is essential for the lung gas exchange function. Disruption of this process results in neonatal death or in severe lung diseases that last into adulthood. We developed live imaging techniques to characterize the mechanisms that control alveolar epithelial cell differentiation. We discovered that mechanical forces generated from the inhalation of amniotic fluid by fetal breathing movements are essential for AT1 cell differentiation. We found that a large subset of alveolar progenitor cells is able to protrude from the airway epithelium toward the mesenchyme in an FGF10/FGFR2 signaling-dependent manner. The cell protrusion process results in enrichment of myosin in the apical region of protruded cells; this myosin prevents these cells from being flattened by mechanical forces, thereby ensuring their AT2 cell fate. Our study demonstrates that mechanical forces and local growth factors synergistically control alveolar epithelial cell differentiation.
Literature context: 47 Phalloidin (catalog #A22287; RRID:AB_2620155) was from Thermo Fisher Scienti
Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression.SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.
The avian nucleus laminaris (NL) is a brainstem nucleus necessary for binaural processing, analogous in structure and function to the mammalian medial superior olive. In chickens (Gallus gallus), NL is a well-studied model system for activity-dependent neural plasticity. Its neurons have bipolar extension of dendrites, which receive segregated inputs from two ears and display rapid and compartment-specific reorganization in response to unilateral changes in auditory input. More recently, fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates local protein translation, has been shown to be enriched in NL dendrites, suggesting its potential role in the structural dynamics of these dendrites. To explore the molecular role of FMRP in this nucleus, we performed proteomic analysis of NL, using micro laser capture and liquid chromatography tandem mass spectrometry. We identified 657 proteins, greatly represented in pathways involved in mitochondria, translation and metabolism, consistent with high levels of activity of NL neurons. Of these, 94 are potential FMRP targets, by comparative analysis with previously proposed FMRP targets in mammals. These proteins are enriched in pathways involved in cellular growth, cellular trafficking and transmembrane transport. Immunocytochemistry verified the dendritic localization of several proteins in NL. Furthermore, we confirmed the direct interaction of FMRP with one candidate, RhoC, by in vitro RNA binding assays. In summary, we provide a database of highly expressed proteins in NL and in particular a list of potential FMRP targets, with the goal of facilitating molecular characterization of FMRP signaling in future studies.
Literature context: o Fisher Scientific Cat# A22287 RRID:AB_2620155 Critical Commercial Assays
Skeletal muscle contraction is mediated by myofibrils, complex multi-molecular scaffolds structured into repeated units, the sarcomeres. Myofibril structure and function have been extensively studied, but the molecular processes regulating its formation within the differentiating muscle cell remain largely unknown. Here we show in zebrafish that genetic interference with the Quaking RNA-binding proteins disrupts the initial steps of myofibril assembly without affecting early muscle differentiation. Using RNA sequencing, we demonstrate that Quaking is required for accumulation of the muscle-specific tropomyosin-3 transcript, tpm3.12. Further functional analyses reveal that Tpm3.12 mediates Quaking control of myofibril formation. Moreover, we identified a Quaking-binding site in the 3' UTR of tpm3.12 transcript, which is required in vivo for tpm3.12 accumulation and myofibril formation. Our work uncovers a Quaking/Tpm3 pathway controlling de novo myofibril assembly. This unexpected developmental role for Tpm3 could be at the origin of muscle defects observed in human congenital myopathies associated with tpm3 mutation.
Literature context: sÂ®, ThermoFisher, ScientificCat#A22287; Lot#1246069Bacterial and Virus
Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host ER, a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the conserved oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.
Literature context: ife Technology Catalog # A22287 RRID:AB_2620155 Aqua-Poly/Mount Polysciences Ca
Theoretical studies suggest that many of the emergent properties associated with multicellular systems arise already in small networks [1, 2]. However, the number of experimental models that can be used to explore collective dynamics in well-defined cell networks is still very limited. Here we focus on collective cell behavior in the female germline cyst in Drosophila melanogaster, a stereotypically wired network of 16 cells that grows by ∼4 orders of magnitude with unequal distribution of volume among its constituents. We quantify multicellular growth with single-cell resolution and show that proximity to the oocyte, as defined on the network, is the principal factor that determines cell size; consequently, cells grow in groups. To rationalize this emergent pattern of cell sizes, we propose a tractable mathematical model that depends on intercellular transport on a cell lineage tree. In addition to correctly predicting the divergent pattern of cell sizes, this model reveals allometric growth of cells within the network, an emergent property of this system and a feature commonly associated with differential growth on an organismal scale .
Literature context: Fisher Scientific Cat# A22287, RRID:AB_262015 Anti-FLAG M2 affinity gel Sigma-
The separation of germline from somatic lineages is fundamental to reproduction and species preservation. Here, we show that Drosophila Germ cell-less (GCL) is a critical component in this process by acting as a switch that turns off a somatic lineage pathway. GCL, a conserved BTB (Broad-complex, Tramtrack, and Bric-a-brac) protein, is a substrate-specific adaptor for Cullin3-RING ubiquitin ligase complex (CRL3GCL). We show that CRL3GCL promotes PGC fate by mediating degradation of Torso, a receptor tyrosine kinase (RTK) and major determinant of somatic cell fate. This mode of RTK degradation does not depend upon receptor activation but is prompted by release of GCL from the nuclear envelope during mitosis. The cell-cycle-dependent change in GCL localization provides spatiotemporal specificity for RTK degradation and sequesters CRL3GCL to prevent it from participating in excessive activities. This precisely orchestrated mechanism of CRL3GCL function and regulation defines cell fate at the single-cell level.
Literature context: er Scientific, catalog #A22287; RRID:AB_2620155) was used for F-actin staining.
Photoreceptor-specific transcription of individual genes collectively constitutes the transcriptional profile that orchestrates the structural and functional characteristics of each photoreceptor type. It is challenging, however, to study the transcriptional specificity of individual photoreceptor genes because each gene's distinct spatiotemporal transcription patterns are determined by the unique interactions between a specific set of transcription factors and the gene's own cis-regulatory elements (CREs), which remain unknown for most of the genes. For example, it is unknown what CREs underlie the zebrafish mpp5bponli (ponli) and crumbs2b (crb2b) apical polarity genes' restrictive transcription in the red, green, and blue (RGB) cones in the retina, but not in other retinal cell types. Here we show that the intronic enhancers of both the ponli and crb2b genes are conserved among teleost species and that they share sequence motifs that are critical for RGB cone-specific transcription. Given their similarities in sequences and functions, we name the ponli and crb2b enhancers collectively rainbow enhancers. Rainbow enhancers may represent a cis-regulatory mechanism to turn on a group of genes that are commonly and restrictively expressed in RGB cones, which largely define the beginning of the color vision pathway.SIGNIFICANCE STATEMENT Dim-light achromatic vision and bright-light color vision are initiated in rod and several types of cone photoreceptors, respectively; these photoreceptors are structurally distinct from each other. In zebrafish, although quite different from rods and UV cones, RGB cones (red, green, and blue cones) are structurally similar and unite into mirror-symmetric pentamers (G-R-B-R-G) by adhesion. This structural commonality and unity suggest that a set of genes is commonly expressed only in RGB cones but not in other cells. Here, we report that the rainbow enhancers activate RGB cone-specific transcription of the ponli and crb2b genes. This study provides a starting point to study how RGB cone-specific transcription defines RGB cones' distinct functions for color vision.
Literature context: n [1:100, AB_2620155 or RRID:AB
Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury.
Literature context: alloidin (RRID:AB_2620155, Thermo Sc
Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.
Literature context: l was from Bethyl Laboratories. F-actin phalloidin 647 (catalog no. A22287) was from M
Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms.
Literature context: # A22287, RRID:AB_2620155; Thermo Fi
Many invertebrates carry out a daily cycle of shedding and rebuilding of the photoreceptor's photosensitive rhabdomeric membranes. The mosquito Aedes aegypti shows a robust response, losing nearly all Aaop1 rhodopsin from the rhabdomeric membranes during the shedding process at dawn. Here, we made use of Aaop1 antibodies capable of distinguishing newly synthesized, glycosylated rhodopsin from mature nonglycosylated rhodopsin to characterize the fate of Aaop1 during the shedding and rebuilding processes. The rhabdomeric rhodopsin is moved into large cytoplasmic vesicles at dawn and is subsequently degraded during the standard 12 h daytime period. The endocytosed rhodopsin is trafficked back to the photosensitive membranes if animals are shifted back to dark conditions during the morning hours. During the daytime period, small vesicles containing newly synthesized and glycosylated Aaop1 rhodopsin accumulate within the cytoplasm. At dusk, these vesicles are lost as the newly synthesized Aaop1 is converted to the nonglycosylated form and deposited in the rhabdomeres. We demonstrate that light acts though a novel signaling pathway to block rhodopsin maturation, thus inhibiting the deglycosylation and rhabdomeric targeting of newly synthesized Aaop1 rhodopsin. Therefore, light controls two cellular processes responsible for the daily renewal of rhodopsin: rhodopsin endocytosis at dawn and inhibition of rhodopsin maturation until dusk. SIGNIFICANCE STATEMENT: Organisms use multiple strategies to maximize visual capabilities in different light conditions. Many invertebrates show a daily cycle of shedding the photoreceptor's rhabdomeric membranes at dawn and rebuilding these during the following night. We show here that the Aedes aegypti mosquito possesses two distinct light-driven cellular signaling processes for modulating rhodopsin content during this cycle. One of these, endocytosis of rhabdomeric rhodopsin, has been described previously. The second, a light-activated cellular pathway acting to inhibit the anterograde movement of newly synthesized rhodopsin, is revealed here for the first time. The discovery of this cellular signaling pathway controlling a G-protein-coupled receptor is of broad interest due to the prominent role of this receptor family across all areas of neuroscience.